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About The Book
Rise in importance of Instrumental Methods of Analysis is due to advancement of medicinal chemistry towards the ultimate target i.e. "better medicines for better life".
This book aims to provide a clear understanding of principles, procedures, limitations & applications in pharmacy of various Spectroscopic and Chromatographic methods. Types of Spectroscopies discussed are UV-visible Spectroscopy, Fluorimetry, IR Spectroscopy, Flame Photometry, Atomic Absorption Spectroscopy and Nepheloturbidometry. Types of Separation Methods(Chromatography) discussed are Adsorption & Partition Column Chromatography, Thin Layer Chromatography, Paper Chromatography, Electrophoresis, Gas Chromatography, High Performance Liquid Chromatography, Ion Exchange Chromatography, Gel Chromatography and Affinity Chromatography. The theoretical & practical knowledge of these instrumental techniques is required by analytical chemists for promoting precision in modern pharmaceutical drug analysis of active pharmaceutical substances as well as secondary pharmaceutical products,namely dosage forms of either single or multi-component formulated product.
The book has been prepared as per the syllabus of Instrumental Methods of Analysis, recommended by PCI for 7th semester of B.Pharm course. The content is formed using simple language in easy-to-understand manner to make the book student-friendly. Each chapter is illustrated with photos, numerous diagrams and schemes of chemical reactions/technical principles. At the end of each chapter, a set of questions and MCQs are also provided.
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1 INTRODUCTION TO SPECTROSCOPY
1.1 Introduction
1.1.1 Wave Property
1.1.2 Wave Parameters and Units
1.1.3. Particle Property
1.2 Electromagnetic Spectrum
1.3 Types of Spectroscopy
Questions
2 ULTRA-VIOLET AND VISIBLE SPECTROSCOPY
2.1 Principle
2.2 Types of Electronic Transitions
2.3 The Chromophore
2.4 The Auxochrome
2.5 Absorption and Intensity Shifts (Spectral shifts) in the UV-Visible Spectroscopy
2.6 Absorption
2.6.1 Laws regarding absorption of light (Beer’s-Lambert’s Law)
2.6.2 Deviations from Beer’s law
2.6.3 Effect of solvent on absorption spectra
2.7 Instrumentation
2.7.1 Source of electromagnetic radiation
2.7.1.1 Visible radiation
2.7.1.2 Ultravoilet radiation
2.7.2 Filters and monochromators
2.7.2.1 Filters
2.7.2.2 Monochromators
2.7.3 Sample cells (cuvettes)
2.7.4 Detectors
2.7.4.1 Barrier layer cell or photo voltaic cell
2.7.4.2 Photo tubes or photoemissive cells
2.7.4.3 Photo Multiplier Tubes (PMT)
2.7.4.4 The photodiode array detector
2.8 Different Types of Instruments
2.9 Applications in Pharmacy
2.9.1 Determination of pKa value
2.9.2 Pharmaceutical quantitative analysis
2.9.2.1 Single component analysis
(a) Using standard absorptivity value (A_1cm^(1%) or ε)
(b) Using a single point and double point standardization method
(c) Using Calibration curve method or multiple standard methods
2.9.2.2 Multicomponent Analysis
(a) Simultaneous equation method
(b) Absorbance ratio method (Q value)
(c) Derivative spectrophotometric method
(d) Difference spectrophotometric method
2.9.3 Spectrophotometric titrations
2.9.4 Preformulation & formulation study
2.9.5 Structural analysis
2.10 Numerical
Questions
3 FLOURIMETRY
3.1 Introduction
3.1.1 Electronic states
3.1.2 Deactivation process
3.2 Theory of Fluorescence
3.3 Types of Fluorescence
3.4 Quantitative Aspects of Fluorimetry
3.5 Factors influencing fluorescence intensity
3.5.1 Concentration
3.5.2 Quantum efficiency (Φ)
3.5.3 Intensity of incident light (I0)
3.5.4 Pathlength (b)
3.5.5 Conjugation
3.5.6 Nature of substituent groups
3.5.7 Rigidity of structures
3. 5.8 Adsorption
3.5.9 Viscosity
3.5.10 Effect of temperature
3.5.11 Oxygen
3.5.12 pH
3.5.13 Photochemical decomposition
3.6 Quenching
3.7 Instrumentation
3.7.1 Source of light
3.7.2 Filters and monochromators
3.7.3 Sample cells
3.7.4 Detectors (Transducer)
3.8 Different Types of Instruments
3.8.1 Single beam filter fluorometer
3.8.2 Double beam filter fluorometer
3.8.3 Single beam spectrofluorometers
3.8.4 Double beam spectrofluorometers
3.9 Advantages and Limitations of Flourimetry
3.10 Applications
3.10.1 Determination of inorganic substances
3.10.2 Determination of organic substances
3.10.3 Pharmaceutical applications
3.10.4 Miscellaneous applications
Questions
4 INFRARED SPECTROSCOPY
4.1 Introduction
4.2 Theory
4.2.1 Types of molecular vibrations
4.2.2 Vibrational frequency
4.2.2.1 Numerical
4.2.2.2 Fundamental modes of vibrations in polyatomic molecules
4.2.2.3 Factors influencing vibrational frequencies
4.3 Instrumentation
4.3.1 IR Radiation Source
4.3.2 Sample handling technique
4.3.2.1 A sampling of gases
4.3.2.2 A sampling of liquids
4.3.2.3 A sampling of solids
4.3.3 Monochromators
4.3.4 Detectors (transducer)
4.3.4.1 Thermal detectors
4.3.4.2 Pyroelectric detector
4.3.4.3 Golay pneumatic detector
4.3.4.4 Photon detectors (photo conducting detectors)
4.4 Different types of instruments
4.4.1 Dispersive spectrophotometer
4.4.2 Fourier transform spectrometers (FTIR)
4.4.2.1 Single beam FTIR spectrometer
4.4.2.2 Double beam FTIR spectrometer
4.4.3 Non-dispersive instruments
4.4.3.1 Filter photometers
4.4.3.2 Photometer without filer
4.5 Application of IR spectroscopy
4.5.1 Identification of substance
4.5.2 Determination of the molecular structure
4.5.3 Studying the progress of reactions
4.5.4 Detection of impurities
4.5.5 Isomerism in organic compounds
4.5.6 Formation of chelates
4.5.7 Analysis of pharmaceutical dosage forms
4.5.8 Miscellaneous
Questions
5 FLAME PHOTOMETRY
5.1 Introduction
5.2 Principle
5.3 Instrumentation
5.3.1 Nebulizer
5.3.2 Burner/Atomizer
5.3.2.1 Total consumption burner
5.3.2.2 Laminar flow (premix) burner
5.3.3 Filter / monochromator
5.3.4 Detector
5.3.5 Recorder/ readout meters
5.4 Different types of instruments
5.4.1 Flame photometer
5.4.2 Flame spectrophotometer
5.5 Interferences
5.5.1 Background absorption
5.5.2 Spectral line interference / cationic interference
5.5.3 Anionic interference/vaporization interference
5.5.4 Ionization interference
5.5.5 Physical interference / viscosity
5.6 Application
5.6.1 Quantitative analysis
5.6.1.1 Direct comparison method
5.6.1.2 Calibration curve method
5.6.1.3 Standard addition method
5.6.1.4 Internal standard method
5.6.2 Assay of pharmaceutical substances (cognate assays)
Questions
6 ATOMIC ABSORPTION SPECTROSCOPY
6.1 Introduction
6.2 Principle
6.3 Instrumentation
6.3.1 Light source
6.3.1.1 Hollow Cathode Lamp (HCL)
6.3.1.2 Electrodeless Discharge Lamps (EDL)
6.3.2 Thermal devices for obtaining atomic aerosols
6.3.2.1 Flame atomization: burner and nebulizer
6.3.2.2 Thermoelectric atomization
6.3.2.3 Chemical vaporization
6.3.3 Monochromator
6.3.4 Detector
6.3.5 Readout device
6.4 Different types of AAS Spectrophotometers
6.4.1 Single-beam atomic absorption spectrophotometer
6.4.2 Double-beam atomic absorption spectrophotometer
6.5 Interferences
6.5.1 Physical interferences
6.5.2 Anionic interferences
6.5.3 Background absorption
6.5.4 Scattering effects
6.5.5 Ionic interferences
6.6 Advantages of AAS over FES
6.7 Disadvantages of AAS
6.8 Applications of AAS
Questions
7 NEPHELOTURBIDOMETRY
7.1 Introduction
7.2 Principle
7.3 Instrumentation
7.3.1 Source of light
7.3.2 Filters and monochromators
7.3.3 Sample cells
7.3.4 Detectors
7.4 Different types of Instruments
7.4.1 Nephelometer
7.4.2 Nephloturbidimeter
7.5 Applications
7.5.1 Analysis of water
7.5.2 Determination of carbon dioxide
7.5.3 Determination of inorganic substances
7.5.4 Cognate Assays
7.5.5. Turbidimetric titrations
Questions
8 INTRODUCTION TO CHROMATOGRAPHY
8.1 Introduction
8.2 Classification of Chromatographic Methods
8.2.1 Based on modes of separation
8.2.2 Based on Principle or mechanism of separation
8.2.3 Based on type of analysis
8.2.4 Based on geometry of stationary phase
8.2.5 Based on development of the chromatogram
8.3 Theory of Chromatography
8.3.1 Plate theory
8.3.2 Rate theory
8.4 Principles underlying chromatographic separations
Questions
9 ADSORPTION AND PARTITION COLUMN CHROMATOGRAPHY
9.1 Introduction
9.1.1 Adsorption column chromatography
9.1.2 Partition column chromatography
9.2 Methodology
9.2.1 Apparatus
9.2.2 Stationary phase or adsorbents
9.2.3 Solvents or mobile phase or eluting agent
9.2.4 Column preparation
9.2.5 Sample loading
9.2.6 Elution or development technique
9.2.7 Detection
9.2.8 Components recovery
9.3 Factors Affecting Column Efficiency
9.4 Advantages
9.5 Disadvantages
9.6 Applications
9.6.1 Adsorption column chromatography
9.6.2 Partition column chromatography
Questions
10 THIN LAYER CHROMATOGRAPHY
10.1 Introduction
10.2 Principle
10.3 Methodology
10.3.1 Thin layer plates
10.3.2 Choice of coating material or adsorbent
10.3.3 Preparation of thin layer on plate
10.3.4 Activation of adsorbent
10.3.5 Purification of silica gel G layer
10.3.6 Sample application
10.3.7 Developing tank
10.3.8 Choice of mobile phase or solvent system
10.3.9 Development technique
(a) Ascending technique
(b) Descending technique
(c) Horizontal and circular development
(d) Multiple or repeated development
10.3.10 Detecting methods
(a) Non specific methods
1. Iodine chamber
2. Charring or sulfuric acid spray reagent
(b) Specific methods
10.3.11 Evaluation of chromatograms
(a) Qualitative evaluation
(b) Quantitative evaluation
1. Direct methods
2. Indirect methods
10.4 Advantages of TLC
10.5 Disadvantages of TLC
10.6 Applications
10.6.1 Test the purity of the sample or drug
10.6.2 For identification and characterization of various drugs
10.6.3 Isolation and separation
10.6.4. In organic chemistry
10.6.5 Biochemical analysis
10.6.6 In food industry
10.6.7 Natural product analysis
10.6.8 Application of TLC in quantitative analysis
10.7 Preparative TLC
10.8 HPTLC
Questions
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11 PAPER CHROMATOGRAPHY
11.1 Introduction
11.1.1 Types of paper chromatography
11.2 Principle
11.3 Methodology
11.3.1 Choice of proper chromatographic technique
11.3.2 Choice of chromatographic paper
11.3.3 Choice of mobile phase
11.3.4 Sample preparation and Spotting
11.3.5 Drying of chromatogram
11.3.6 Visualization
(a) Non-specific methods
1. UV chamber for fluorescent compounds
2. Iodine chamber method
(b) Specific methods
11.3.7 Evaluation of chromatogram
(a) Qualitative evaluation
(b) Quantitative evaluation
11.3.8 Development techniques
(a) Ascending chromatography
(b) Descending chromatography
(c) Ascending-descending chromatography
(d) Radial paper chromatography
(e) Two dimensional Chromatography
11.4. Advantages of paper chromatography
11.5 Disadvantages of paper chromatography
11.6 Precautions to be taken care while performing paper chromatography
11.7 Applications of paper chromatography
11.7.1 Separation of amino acid
11.7.2 Separation of mixture of sugars
11.7.3 Identification of drugs
11.7.4 Identification of impurities
11.7.5 Identification of related compounds
11.5.6 For identification decomposition products
11.5.7 For analysis of metabolites of drugs in blood, urine
Questions
12 ELECTROPHORESIS
12.1 Introduction
12.2 Factors Affecting Electrophoretic Mobility
12.2.1 Sample/Solute property
(a) Charge
(b) Size
(c) Shape
12.2.2 The environment or the laboratory system
(a) Electric field
(b) Buffer
(c) Supporting medium
12.3 Techniques of Electrophoresis
12.3.1 Moving boundary or free boundary electrophoresis
12.3.2 Zone electrophoresis/electrochromatography
12.4 Paper Electrophoresis
12.4.1 Components of paper electrophoresis
(a) Paper
(b) Electrolytes or buffer
(c) Electrodes
(d) Source of current
12.4.2 Types of paper electrophoresis
(a) High Voltage paper electrophoresis
(b) Low Voltage paper electrophoresis
12.4.3 Methodology
12.4.4 Advantages of paper electrophoresis
12.4.5 Disadvantages of paper electrophoresis
12.4.6 Applications of paper electrophoresis
12.5 Gel Electrophoresis
12.5.1 Methodology
12.5.1.1 Gel
(a) Types of Gels
(b) Preparation of Gels
12.5.1.2 Sample application and visualization
12.5.2 Types of gel electrophoresis
12.5.3 Advantages of gel electrophoresis
12.5.4 Disadvantages of gel electrophoresis
12.5.5 Applications of gel electrophoresis
12.6 Capillary Electrophoresis
12.6.1 Techniques of Capillary Electrophoresis
(a) Capillary Zone Electrophoresis (CZE)
(b) Capillary Gel Electrophoresis (CGE)
(c) Capillary Isotachophoresis (CIE)
(d) Electrokinetic Capillary Chromatography (MECC)
12.6.2 Methodology
12.6.3 Variations in capillary electrophoresis
12.6.4 Advantages of capillary electrophoresis
12.6.5 Disadvantages of capillary electrophoresis
12.6.6 Applications of capillary electrophoresis
Questions
13 GAS CHROMATOGRAPHY
13.1 Introduction
13.2 Theory
13.3 Instrumentation
13.3.1 Carrier gas
13.3.2 Flow regulator or flow meter
13.3.3 Injection Devices
13.3.4 Columns
(a) According to nature
1. Packed column
2. Capillary column / open tubular column / golay column
(b) According to use
1. Analytical column
2. Preparative column
13.3.5 Temperature control device (temperature programming)
13.3.6 Detectors
(a) Flame Ionization Detector (FID)
(b) Thermal Conductivity Detector (TCD)
(c) Electron Capture Detector (ECD)
(d) Thermionic Detector (TID)
(e) Electrolytic Conductivity Detector
(f) Photoionization Detector (PID)
(g) Flame photometric detector (FPD)
(h) Mass spectrometer (MS) Detector
13.3.7 Recorder and integrator
13.4 Derivatization
13.4.1 Ideal Characteristic of derivatization
13.4.2 Limitations
13.4.3 Types of Derivatization
(a) Alkylation
(b) Silylation
(c) Acylation
(d) Chiral derivatization
13.5 Applications
(a) Qualitative analysis
1. Checking the purity of compound
2. Presence of impurities
(b) Quantitative analysis
1. Direct comparison method
2. Calibration curve method
3. Internal standard method
(c) Multicomponent analysis
(d) Food Product analysis
(e) Inorganic compounds
(f) Environmental studies
(g) Forensic science
Questions
14 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
14.1 Introduction
14.2 Principle
14.3 Theory
14.4 Chromatographic Parameters
14.5 Instrumentation
14.5.1 Solvent reservoir and mixing system
14.5.2 High pressure pump
(a) Types of HPLC pumps
1. Reciprocating pumps
2. Displacement or syringe pumm
3. Pneumatic or constant pressure pump
14.5.3 Sample inlet or injection system
(a) Septum Injector
(b) Loop valve type or rheodyne injectors
(c) Stopped-flow injection
14.5.4 Columns
(a) Analytical columns
1. Standard column
2. Radial compression columns
3. Narrow bore columns
4. Fast, Short Columns
(b) Guard columns
(c) Column Packing
(i) The materials used for column packing
1. Totally porous
2. Pellicular or superficially porous
3. Bonded Phases
(ii) HPLC stationary phases
1. Octadecyl silica (ODS or C18)
2. Size exclusion and ion-exchange stationary phases
3. Chiral stationary phases
(d) Temperature Control
14.5.5 Detector and recording unit
1. Refractive index detectors (RI detectors)
2. Conductivity detectors
3. UV-visible spectrophotometric detectors
4. Infrared absorption detectors
5. Fluorescence detectors
6. Electrochemical detectors
7. Evaporative Light Scattering Detectors (ELSD)
8. Mass Detectors
(a) Particle Beam Ionization
(b) Thermospray Interface
(c) Electrospray Ionization
(d) Atmospheric pressure chemical Ionization (APCI)
14.6 Advantages of HPLC
14.7 Applications of HPLC
(a) For Clinical diagnosis of diseases and disorders
(b) In scientific research for discovery
(c) In pharmaceutical labs for analysis
(d) In the food industry for quality control
(e) For standards control by government
(f) Testing for Pesticides Residue
(g) Forensic application
Questions
15 ION EXCHANGE CHROMATOGRAPHY
15.1 Introduction
15.2 Principle
15.3 Classification of ion exchange resins
15.3.1 According to Chemical nature
15.3.2 According to source
15.3.3 According to structure
15.4 Properties of ion exchange resins
(a) Physical Properties
1. Particle size and porosity
2. Cross linking and swelling
3. Regeneration
(b) Chemical Properties
15.5 Mechanism of ion exchange process
15.6 Methodology of ion exchange chromatography
15.6.1 Column material and dimensions
15.6.2 Type of ion exchange resin and physical characteristics
15.6.3 Mobile phase
15.6.4 Packaging of column
15.6.5 Application of sample
15.6.6 Elution
15.6.7 Analysis of elutent
15.7 Factors affecting ion exchange separation
(a) Nature and properties of ion exchange resin
(b) Nature of exchanging ions
15.8 Applications
1. Demineralization of water
2. Softening of water
3. Separation of sugars
4. Purification of organic compounds
5. Separation of amino acids
6. Purification and recovery of pharmaceuticals
7. Biochemical separation
8. Medicinal importance
9. Ion exchange column in HPLC
Questions
16 GEL CHROMATOGRAPHY
16.1 Introduction
16.2 Theory
16.3 Instrumentation
16.3.1 Column
16.3.2 Stationary phase
16.3.3 Mobile phase
16.3.4 Elution
16.3.5 Detector
16.4 Factors affecting ressolution in gel chromatography
16.5 Steps of gel chromatography
16.6 Advantages of gel chromatography
16.7 Applications
1. Analysis of synthetic polymers or bio-polymers
2. Separation of proteins in mixture
3. Molecular weight determination
4. Molecular weight distribution analysis
5. Purification of macromolecules, proteins, enzymes, amino acids,
polysaccharides
6. Desalting of proteins
Questions
17 AFFINITY CHROMATOGRAPHY
17.1 Introduction
17.2 Theory
17.3 Instrumentation
17.3.1 Steps of affinity chromatography
1. Application of sample
2. Washing
3. Elution
4. Regenration
17.3.2 Stationary phase for affinity chromatography
(a) Affinity supports (matrix)
(b) Spacer arm
(c) Affinity ligand
1. Mono specific ligands
2. Group specific ligands
17.4 Applications
Questions
APPENDICES
I Woodward-Fisher Rules
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